Mitochondrial oxidative phosphorylation is the main system to efficiently supplying ATP for the eukaryotic cell. Electron transfer through the respiratory chain concomitantly translocates protons across the inner membrane at three coupling sites (complex I, III, and IV). Complex I (CI, NADH:ubiquinone oxidoreductase) provides ~40% of proton-motive force for the ATP synthesis, while CI is one of the major sites of reactive oxygen species (ROS) generation and is extremely vulnerable to oxidative stress. Thus CI dysfunction is implicated in a variety of mitochondrial diseases including heart failure, type 2 diabetes, and neurodegenerative diseases such as Parkinson's disease. Therefore, the elucidation of CI mechanisms is crucial for understanding these diseases and developing therapeutic strategies. However, CI has been one of the most challenging molecules to study its mechanisms and functions, because of the gigantic size (~900 kDa) and complexity of 45 different subunits and several cofactors including as many as 8 iron-sulfur clusters. The central fundamental question of how electron transfer is linked to vectorial proton translocation in CI still remains unanswered even 30 years since Peter Mitchell won the Nobel prize for his chemiosmotic theory. The long-term goal of this proposal is to elucidate the redox-coupled proton (H+) pump mechanism in CI. Previously, the PI made significant findings from a photoaffinity labeling study with fenpyroximate (a potent CI inhibitor that ND5, a transporter module membrane subunit, is involved in both ubiquinone(UQ)-binding and H+ translocation. This led us to start mutational study of the NuoL subunit (E. coli ND5 homolog). It has been predicted that in CI, redox chemistry drives proton translocation via an indirect (conformation-driven) coupling mechanism, but there was no testable details. We recently found the possibility that two major but tightly coupled functions, electron transfer (ET) and proton (H+) pump, could be decoupled by novel mutations in ND5. This strongly suggests that CI operates an indirect coupling mechanism. We hypothesize that NuoL(ND5) is the key player in the indirect conformation-driven coupling mechanism in CI. Specific aims are: Aim1. Analyze the relationship between ET and H+ pumping activities in a series of novel NuoL mutants. Aim2. Elucidate conformational changes important for the indirect H+ pump coupling mechanism. Aim3. Investigate the involvement of other transporter subunits NuoM(ND4) and NuoN(ND2) in indirect coupling. This project will provide a molecular level of understanding of indirect (conformation-driven) coupling process in CI.